Coated plant virus imaging agents

ABSTRACT

An imaging nanoparticle comprising a plant virus particle having an interior surface and an exterior surface, an imaging agent that is linked to the interior and/or exterior surface, and a layer of biocompatible mineral such as silica coated over the exterior surface, is described. The imaging nanoparticle can be used in method of generating an image of a tissue region of a subject, by administering to the subject a diagnostically effective amount of an imaging nanoparticle and generating an image of the tissue region of the subject to which the imaging nanoparticle has been distributed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/033,297, filed Aug. 5, 2014, which is incorporated herein byreference

GOVERNMENT FUNDING

The present invention was supported by a grant from the National ScienceFoundation (CMMI NM 1333651) and the National Institutes of Health (NIHR21HL121130). The Government has certain rights in this invention.

BACKGROUND

Molecular imaging facilitates the early detection of disease, allowsrisk stratification, disease monitoring, longitudinal imaging andtreatment follow up. A variety of imaging modalities have beendeveloped, including positron electron tomography (PET), computedtomography (CT), and magnetic resonance imaging (MRI). The latter isgaining popularity because of its excellent soft tissue contrast,spatial resolution and penetration depth, and because the non-ionizingradiation is safer for repeated imaging sessions. However, MRI has a lowsensitivity to contrast-enhancement agents, which provide importantinformation about molecular features in vivo. Nanoparticles are idealplatforms for the development of better contrast-enhancement agentsbecause they can carry large payloads, they can be modified withtargeting ligands to confer molecular specificity and their structureenhances ionic relaxivity.

Several nanoparticle-based MRI contrast agents have been described,including nanoemulsions, dendrimers, silica and gold nanoparticles, andviral nanoparticles (VNPs). Bruckman et al., Nanotechnology. 2013;24(46):462001. Nanoparticles increase the longitudinal relaxivity(positive contrast, R₁) by reducing the molecular tumbling rate (τ_(R))of chelated paramagnetic ions such as Gd following surface conjugation.Caravan et al., 2009; 4(2):89-100. In theory, free chelated Gd ions witha relaxivity of ˜5 mM⁻¹ s⁻¹ can achieve relaxivities of up to 80 mM⁻¹s⁻¹ at 1.5 T, the common mode of MRI used in the clinic. This is basedon the optimization of properties such as particle stiffness, bulk wateraccessibility and the chelating molecule, although experimentally itremains challenging to achieve such high values.

The inventors have focused the development of VNPs for medicalapplications because the manufacture of such proteinaceous nanoparticlesin a variety of shapes and sizes is highly reproducible and scalable,and the particles themselves are amenable to functionalization usingsynthetic biology, genetic engineering and bioconjugation chemistry. VanKan-Davelaar et al., British Journal of Pharmacology. 2014;171(17):4001-4009. Several VNP-based MRI contrast agents have beendescribed, including the icosahedral plant viruses Cowpea mosaic virus(CPMV) (Prasuhn et al., Chemical Communications. 2007(12):1269), Cowpeachlorotic mottle virus (CCMV) (Liepold et al., Magnetic Resonance inMedicine. 2007; 58(5):871-9), bacteriophages P22, MS2 and Qβ, and theplant virus Tobacco mosaic virus (TMV), which naturally occurs as rodsbut can also be produced as spheres. Bruckman et al., Journal ofMaterials Chemistry B. 2013; 1(10):1482A.

few recent articles discuss the in vivo performance of theseprotein-based MRI contrast agents. Min et al., Biomacromolecules. 2013;14(7):2332-9. For example, the inventors recently showed that TMVparticles can be employed to image the molecular features ofatherosclerotic plaques using a vascular cell adhesion molecule(VCAM-1)-targeted Gd(DOTA)-loaded probe. Bruckman et al., Nano Letters.2014; 14(3):1551-8. The T₁ relaxivity of this nanoparticle was ˜15 mM⁻¹s¹, yielding a per particle relaxivity of 35,000 mM⁻¹ s¹ at 60 MHz, thusallowing the imaging of molecular features in vivo at submicromolardoses of Gd(DOTA). However, there remains a need for imaging agents withimproved performance, such as increased sensitivity and decreasedimmunogenicity.

SUMMARY

The inventors have investigated the materials and biological propertiesof TMV-based MRI contrast agents, specifically to develop probes formacrophage imaging. The active or passive targeting of immune cells is auseful strategy to investigate the cellular components involved indisease progression associated with inflammation. Macrophage imaging wasstudied as a function of contrast agent shape and surface coating.Protein-based nanoparticles (TMV rods and TMV spheres) were mineralizedwith silica coatings.

The inventors chose silica as a coating material because it isbiologically inert and coating techniques are well established. Tarn etal., Accounts of Chemical Research. 2013:46(3):792-801. For example,silica mineralization has been used to improve the biocompatibility ofnanoparticles based on gold (Lee et al., Toxicology Letters. 2012;209(1):51-7), iron oxide (Singh et al., Journal of Biomedical MaterialsResearch Part A. 2012; 100A(7):1734-42) and quantum dots (Durgadas etal., Biomaterials. 2012; 33(27):6420-9). The inventors hypothesized thatthe silica coating would maintain high relaxivities, while providing ameans for antibody evasion. Research indicates that TMV-specificantibodies are prevalent in the population due to presence of TMV infood and cigarettes. Liu et al., PLoS ONE. 2013; 8(4):e60621. The silicashell was therefore investigated to see if it would protect TMV and SNPfrom recognition by TMV-specific antibodies. This is an important goalfor potential clinical application to prevent premature clearance of thecontrast agent and maintain stable and reproducible pharmacokinetics forrepeated imaging sessions.

In one aspect, the present invention provides an imaging nanoparticle,comprising a plant virus particle having an interior surface and anexterior surface, an imaging agent that is linked to the interior and/orexterior surface, and a layer of biocompatible mineral coated over theexterior surface. In some embodiments, the biocompatible mineral issilica, with in further embodiments the plant virus is a rod-shapedvirus particle.

Another aspect of the present invention provides a method of generatingan image of a tissue region of a subject. The method includesadministering to the subject a diagnostically effective amount of animaging nanoparticle, comprising a plant virus particle having aninterior surface and an exterior surface, an imaging agent that islinked to the interior and/or exterior surface, and a layer ofbiocompatible mineral coated over the exterior surface, and generatingan image of the tissue region of the subject to which the imagingnanoparticle has been distributed. In some embodiments, thebiocompatible mineral is silica, with in further embodiments the plantvirus is a rod-shaped virus particle.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thefollowing drawings.

FIG. 1 (A, B) provides a A) the bioconjugation and mineralization schemewhich produces Gd(DOTA)-loaded and silica-coated TMV rods and spheres;and B) TEM and SEM imaging of TMV-based contrast agents. Scale bars=100nm (TEM, black) or 500 nm (SEM, white).

FIG. 2 (A-F) provides a table (A) and accompanying (B) bar graph showingGd loading per particle, relaxivity per Gd and per particle (in mM⁻¹ s⁻¹at 60 MHz). The lower panels show relaxivity curves for (C) iGd-TMV, (D)eGd-TMV, (E) Gd-SNP, and (F) unmodified TMV. One curve is forpre-mineralized (native) particles, while the other curves arepost-mineralized particles. TMVlys shows the relaxivity curves forunlabeled TMVlys at virus concentrations matching values for iGd-TMVcurves. *Gd per SNP calculated based on their size/volume relationships.

FIG. 3 (A-D) provides graphs and images showing TMV, SNP, Si-TMV andSi-SNP interactions with RAW 264.7 cells 1, 3, and 8 h after exposureusing fluorescent TMV and SNP formulations, with (A) providinghistograms from flow cytometry studies; (B) providing mean intensityplotted versus time and per particle formulation as a quantitativemeasure of cell interactions; (C) providing MRI phantom images of RAW264.7 cell pellets 8 h after binding with TMV, SNP, Si-TMV and Si-SNP.Gd(DOTA)-labeled TMV and SNP formulations were incubated with RAW 264.7cells for 8 h, then cells were washed and pelleted prior to obtainingMRI images using a 7.0 T (300 MHz) MRI (Bruker BioSpec 70/30USR). Thearrow indicates the cell pellets, a positive signal shows as brightpixels; and (D) providing a graph showing cell interactions werequantified by contrast-to-noise (CNR) ratio of the MRI phantom image(cell pellets vs. medium).

FIG. 4 (A-E) provides images showing the Binding of gold-labeledanti-TMV antibodies to (A) TMV, (B) SNP, (C) Si-TMV, (D) Si-SNP, and (E)a mix of TMV and Si-TMV. Scale bars=100 nm.

FIG. 5 provides a schematic diagram showing the bioconjugation of TMVrods.

FIG. 6 provides a graph showing the MALDI-TOF MS spectra for modifiedTMV particles. Peak assignments: native TMV=17,534 m/z; iGd-TMV=17,639m/z (alkyne-modified) and 18,390 m/z (Gd(DOTA) modified); eGd-TMV=17,534m/z (unmodified), 17,729 m/z (alkyne modified), and 18,318 m/z (Gd(DOTA)modified).

FIG. 7 provides a graph showing the electron dispersion spectra of TMVbefore and after mineralization confirming the presence of silica aftermineralization.

FIG. 8 provides a table and graph showing the relaxivity of TMV rodsloaded with varying amounts of Gd(DOTA), where eq=molar equivalents.Measurements at 60 MHz. Relaxivity values in mM⁻¹ s⁻¹.

FIG. 9 (A-C) provides high-resolution TEM images of (A) iGd-TMV-Si, (B)eGd-TMV-Si, and (C) Gd-SNP-Si showing dense silica coat. Scale bar=25nm.

FIG. 10 (A-C) provides graphs and images; top panels: MRI phantom imagesof RAW 264.7 cell pellets 8 h after binding with Gd-TMV, Gd-SNP,Si-Gd-TMV and Si-Gd-SNP. Gd(DOTA)-labeled TMV and SNP formulations wereincubated with RAW 264.7 cells for 8 h, then cells were washed andpelleted prior to obtaining MRI images using a 7.0 T (300 MHz) MRI(Bruker BioSpec 70/30USR). In A and B, 1,000,000 cells were incubatedwith (A) 250,000 VNP per cell and (B) 2,500,000 VNP per cell. In panelC, 5,000,000 cells were incubated with 1,000,000 VNP per cell. Bottompanels: Cell interactions were quantified by contrast-to-noise (CNR)ratio of the MRI phantom image (cell pellets vs. medium).

DETAILED DESCRIPTION

Imaging nanoparticles comprising a plant virus particle having aninterior surface and an exterior surface, an imaging agent that islinked to the interior and/or exterior surface, and a layer ofbiocompatible mineral such as silica coated over the exterior surfaceare described. The imaging nanoparticles can be used in a method ofgenerating an image of a tissue region of a subject such as a tumor oratherosclerotic tissue by administering the imaging nanoparticles to thesubject and generating an image of the tissue region of the subject towhich the imaging nanoparticles have distributed.

Definitions

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural references unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes a combination of two or morecells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or 110%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

“Image” or “imaging” refers to a procedure that produces a picture of anarea of the body, for example, organs, bones, tissues, or blood.

A “subject,” as used herein, can be any animal, and may also be referredto as the patient. Preferably the subject is a vertebrate animal, andmore preferably the subject is a mammal, such as a domesticated farmanimal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In someembodiments, the subject is a human.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject for the methodsdescribed herein, without unduly deleterious side effects.

As used herein, the term “relaxation time” refers to the time requiredfor a nucleus which has undergone a transition into a higher energystate to return to the energy state from which it was initially excited.Regarding bulk phenomena, the term “relaxation time” refers to the timerequired for a sample of nuclei, the Boltzmann distribution of which hasbeen perturbed by the application of energy, to reestablish theBoltzmann distribution. The relaxation times are commonly denoted T₁ andT₂. T₁ is referred to as the longitudinal relaxation time and T₂ isreferred to as the transverse relaxation time. As used herein, the term“relaxation time” refers to the above-described relaxation times eithertogether or in the alternative. An exhaustive treatise on nuclearrelaxation is available in Banci, L, et al. Nuclear and ElectronRelaxation, VCH, Weinheim, 1991, which is herein incorporated byreference.

As used herein, the term “diagnostically effective amount” refers to anamount of contrast agent that is sufficient to enable imaging of thecontrast agent in cells, tissues, or organisms using imaging equipment.

As used herein, a protein such as an antibody “specifically binds” whenthe antibody preferentially binds a target structure, or subunitthereof, but binds to a substantially lesser degree or does not bind toa biological molecule that is not a target structure. Antibodies thatspecifically bind to a target structure, or subunit thereof, do notcross-react with biological molecules that are outside the targetstructure family.

“Targeting,” as used herein, refers to the ability of an imagingnanoparticle to be delivered to and preferentially accumulate in thetarget tissue in a subject.

“Biocompatible” as used herein, refers to any material that does notcause injury or death to the animal or induce an adverse reaction in ananimal when placed in intimate contact with the animal's tissues.Adverse reactions include for example inflammation, infection, fibrotictissue formation, cell death, or thrombosis. The terms “biocompatible”and “biocompatibility” when used herein are art-recognized and mean thatthe material is neither itself toxic to a subject, nor degrades (if itdegrades) at a rate that produces byproducts (e.g., monomeric oroligomeric subunits or other byproducts) at toxic concentrations, doesnot cause prolonged inflammation or irritation, or does not induce morethan a basal immune reaction in the host.

In one aspect, the present invention provides an imaging nanoparticle.The imaging nanoparticle comprises a plant virus particle having aninterior surface and an exterior surface, an imaging agent that islinked to the interior and/or exterior surface, and a layer ofbiocompatible mineral coated over the exterior surface.

Plant Viruses

The imaging nanoparticles of the present invention are based on plantvirus particles. Plant virus particles preferably grow in plants, andhave the advantages of being readily cultivated, and are unlikely tocause infection when used in vivo in a subject. Plant virus particlesare categorized based on their source and structure. In variousembodiments, virus particles having an icosahedral, filamentous, orrod-shaped structure can be used. Preferably, the virus particles usedare non-enveloped virus particles. Examples of icosahedral plant virusesinclude cowpea mosaic virus, brome mosaic virus, cowpea chlorotic mottlevirus, etc. Use of filamentous or rod-shaped plant virus particles ispreferred, in part as a result of the proclivity of these viralparticles to be taken up by diseased tissue.

A filamentous plant virus is a virus that primarily infects plants andhas a non-enveloped filamentous structure. A filamentous structure is along, thin virion that has a filament-like or rod-like shape that ismuch longer than it is wide and therefore has a high-aspect ratio. Forexample, Alphaflexiviridae have a length of about 470 to about 800 nm,and a diameter of about 12-13 nm. Filament-like virus particles areflexible in addition to being long and thin, and therefore someembodiments of the invention are directed to use of a flexiblefilamentous plant virus. As described herein, use of filamentous plantviruses provides the advantages of improved tumor targeting andpenetration. Embodiments of the invention can deliver about 10%, about20%, about 30%, about 40%, or even about 50% or more of the injecteddose to tumor tissue.

In some embodiments, the filamentous plant virus belongs to a specificvirus family, genus, or species. For example, in some embodiments, thefilamentous plant virus belongs to the Alphaflexiviridae family. TheAlphaflexiviridae family includes the genus Allexivirus, Botrexvirus,Lolavirus, Mandarivirus, Potexvirus, and Sclerodamavirus. In someembodiments, the filamentous plant virus belongs to the genusPotexvirus. In further embodiments, the filamentous plant virus belongsto the Potato Virus X species.

In some embodiments, the imaging nanoparticle is based on a rod-shapedplant virus. A rod-shaped plant virus is a virus that primarily infectsplants, is non-enveloped, and is shaped as a rigid helical rod with ahelical symmetry. Rod shaped viruses also include a central canal.Rod-shaped plant virus particles are distinguished from filamentousplant virus particles as a result of being inflexible, shorter, andthicker in diameter. For example, Virgaviridae have a length of about200 to about 400 nm, and a diameter of about 15-25 nm. Virgaviridae haveother characteristics, such as having a single-stranded RNA positivesense genome with a 3′-tRNA like structure and no polyA tail, and coatproteins of 19-24 kilodaltons.

In some embodiments, the rod-shaped plant virus belongs to a specificvirus family, genus, or species. For example, in some embodiments, therod-shaped plant virus belongs to the Virgaviridae family. TheVirgaviridae family includes the genus Furovirus, Hordevirus,Pecluvirus, Pomovirus, Tobamovirus, and Tobravirus. In some embodiments,the rod-shaped plant virus belongs to the genus Tobamovirus. In furtherembodiments, the rod-shaped plant virus belongs to the tobacco mosaicvirus species. The tobacco mosaic virus has a capsid made from 2130molecules of coat protein and one molecule of genomic single strand RNA6400 bases long. The coat protein self-assembles into the rod likehelical structure (16.3 proteins per helix turn) around the RNA whichforms a hairpin loop structure. The protein monomer consists of 158amino acids which are assembled into four main alpha-helices, which arejoined by a prominent loop proximal to the axis of the virion. Virionsare ˜300 nm in length and ˜18 nm in diameter. Negatively stainedelectron microphotographs show a distinct inner channel of ˜4 nm.

In some embodiments, the plant virus is an icosahedral plant virus.Examples of icosahedral plant viruses include the virus familiesGeminiviridae, Luteoviridae, Bromoviridae, Phycodnaviridae, andPicornaviridae. In some embodiments, the icosahedral plan virus is fromthe family Picornaviridae. Plant picornaviruses are relatively small,non-enveloped, positive-stranded RNA viruses with an icosahedral capsid.Plant picornaviruses have a number of additional properties thatdistinguish them from other picornaviruses, and are categorized as thesubfamily secoviridae. In some embodiments, the virus particles areselected from the Comovirinae virus subfamily. Examples of viruses fromthe Comovirinae subfamily include Cowpea mosaic virus, Broad bean wiltvirus 1, and Tobacco ringspot virus. In a further embodiment, the virusparticles are from the Genus Comovirus. A preferred example of aComovirus is the cowpea mosaic virus particles.

Spherical Nanoparticles

Rod-shaped plant virus particles can be combined with other rod-shapedplant virus particles by means of a thermal transition to form anRNA-free spherical nanoparticle (SNP), also referred to herein as aspherical nanoparticle imaging platform. A spherical nanoparticleimaging platform is a spherical arrangement of the coat proteins of aplurality of rod-shaped plant virus particles linked to an imaging agenton an interior surface of the virus particle, formed by thermaltransition of the rod-shaped virus particles. The SNPs can be formedfrom rod-shaped plant virus particles bearing imaging agents linked tothe interior surface of the rod-shaped plant virus particles. SNPs canbe labeled with suitable chemicals prior or post thermal transition; forexample, NHS-based chemistries allow one to conjugate functionalmolecules to SNPs post thermal transition; the SNPs are stable andremain structurally sound after chemical modification. The SNPsincluding imaging agent can be formed from rod-shaped plant virusparticles (e.g., TMV virus particles) by briefly heating the rod-shapedplant virus particles labeled with imaging agent on an interior surfaceof the virus particle. For example, the rod-shaped plant virus particlescan be induced to undergo a thermal transition into SNPs by heating atabout 96° C. for about 10 to about 20 seconds. Examples of suitablerod-shaped virus particles include Virgaviridae virus particles andtobacco mosaic virus particles. Any of the imaging agents describedherein can be used with the spherical nanoparticles. In someembodiments, the imaging agent is a chelated lanthanide such asgadolinium.

The SNPs are formed from the coat proteins of one or more individualrod-shaped plant virus particles. In various embodiments, the SNP can beformed from about 1 to 10 virus particles, from about 10 to about 20virus particles, from about 20 to about 30 virus particles, from about30 to about 40 virus particles, or from about 40 to about 50 virusparticles. Depending on the nature of the coat proteins, the number ofvirus particles incorporated, and the virus particle concentration inthe solution in which the thermal transition occurs, the sphericalnanoparticles can also vary in size. In some embodiments, the SNPs havea size from about 50 nm to about 800 nm. In further embodiments, theSNPs have a size from about 100 to about 300 nm, or from about 150 toabout 200 nm.

Spherical nanoparticles including imaging agents such as chelatedgadolinium provide several advantages. First, SNPs can include a highper-particle concentration of imaging agent. For example, SNPs caninclude from about 3,000 to about 30,000 imaging agents per sphericalnanoparticle, with about 20,000 to about 30,000 imaging agent moleculesper spherical nanoparticle in some embodiments. In addition, for MRIimaging agents such as chelated lanthanides, the SNPs including imagingagents can also exhibit very high relaxivity per particle. For example,SNPs including lanthanide imaging agents can exhibit a T₁ relaxivity perparticle from about 10,000 mM⁻¹ s⁻¹ to about 500,000 mM⁻¹ s⁻¹ at 60 MHz,with about 350,000 mM⁻¹ s⁻¹ to about 450,000 mM⁻¹ s⁻¹ at 60 MHz in someembodiments. Finally, SNPs are more rapidly cleared from the body, whichcan be advantageous with imaging agents that may have increased adverseside effects when they persist within the subject after imaging.

Biocompatible Minerals

A variety of different biocompatible minerals can be used to coat thesurface of the plant virus particles. Examples of biocompatible mineralsinclude silicates, graphene, mineral trioxide, calcium phosphate, ironoxide, and carbonates. In some embodiments, the biocompatible mineral issilica (i.e., silicon dioxide). Other examples of silicates includebiosilicates and calcium silicate.

A layer of biocompatible mineral coated over the exterior surface of thevirus particle. The biocompatible mineral may cover all of the exteriorsurface of the virus particle, or it may cover a significant portion ofthe exterior of the virus particle. For example, the biocompatiblemineral may cover at least about 50%, 60%, 70%, 80%, or at least about90% of the surface of the exterior of the virus particle. A wide varietyof different biocompatible minerals can be coated onto virus particlesusing eletrophoretic deposition. Boccaccini et al., J R Soc Interface, 7Suppl 5:S581-613 (2010). Alternately, the virus particle can be coatedusing evaporation induced self-assembly. Tarn et al., Acc Chem. Res.2013, 46, 792-801. The layer of biocompatible mineral should be thin toavoid adding unnecessary bulk to the virus particles. In someembodiments, the layer has a thickness from about 1 to about 100nanometers, while in other embodiments the layer has a thickness fromabout 20 to about 50 nanometers.

Imaging Agents

The plant virus particle is modified to carry an imaging agent. Examplesof imaging agents include fluorescent compounds, radioactive isotopes,and MRI contrast agents. For example, in some embodiments, the imagingagent is a fluorescent molecule for fluorescent imaging. The detectablegroup can be any material having a detectable physical or chemicalproperty. Such imaging agents have been well-developed in the field offluorescent imaging, magnetic resonance imaging, positive emissiontomography, or immunoassays and, in general, most any imaging agentuseful in such methods can be applied to the present invention. Thus, animaging agent is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful imaging agents in the present invention includemagnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, AlexaFluor555, Texas red, rhodamine, and the like),radiolabels (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹²¹I, ¹¹²In, ⁹⁹mTc), otherimaging agents such as microbubbles (for ultrasound imaging), ¹⁸F, ¹¹C,¹⁵O, (for Positron emission tomography), ⁹⁹mTC, ¹¹¹In (for single photonemission tomography), and chelated lanthanides such as terbium,gadolinium, and europium (e.g., chelated gadolinium) or iron (formagnetic resonance imaging). The choice of imaging agent depends on thesensitivity required, the ease of conjugation with the compound,stability requirements, available instrumentation, and disposalprovisions.

In some embodiments, the imaging agent is a magnetic resonance imagingagent. Disease detection using MRI is often difficult because areas ofdisease have similar signal intensity compared to surrounding healthytissue. In the case of magnetic resonance imaging, the imaging agent canalso be referred to as a contrast agent. Lanthanide elements are knownto be useful as contrast agents. The lanthanide chemical elementscomprises the fifteen metallic chemical elements with atomic numbers 57through 71, and include lanthanum, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, and lutetium. Preferred lanthanidesinclude europium, gadolinium, and terbium. In order to more readilyhandle these rare earth metals, the lanthanides are preferably chelated.In some embodiments, the lanthanide selected for use as a contrast agentis gadolinium, or more specifically gadolinium (III).

Contrast agents are used to enhance the differentiation between tissueregions in order to better image the tissue. The ionic relaxivity rateof a contrast agent describes its capacity for contrast enhancement. Therelaxivity rate can be affected by a number of factors, including theuse of a chelating agent. Unless indicated otherwise, all relaxivitymeasurements described herein are at 60 MHz, which is the field strengthat which the relaxivity was typically measured. A clinical 3.0 Teslamagnet measures at that field strength. However, it should be noted thatpreclinical imaging is often done at higher magnetic field strength, andthat the relaxivity can change with the field strength. The relaxivityrate per plant virus particle can also be increased by increasing thenumber of agent molecules that are linked to the virus particle. Plantvirus particles of the invention that have been chemically modified toinclude contrast agents can exhibit relaxivity rates from about 10,000to about 40,000 mM⁻¹ S⁻¹. In some embodiments, the virus particlesbearing contrast agents exhibit T₁ relaxivity rates of at least about10,000 mM⁻¹ S⁻¹, about 20,000 mM⁻¹ S⁻¹, about 25,000 mM⁻¹ S⁻¹, about30,000 mM⁻¹ S⁻¹, about 35,000 mM⁻¹ S⁻¹, and about 40,000 mM⁻¹ S⁻¹ at 60MHz.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the virusparticle. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands.

Conjugation of Imaging Agents

The invention makes use of a plant virus particle that has been modifiedto carry an imaging agent. Including an imaging agent allows the virusparticle to serve as a platform for the imaging agent. A plant virusparticle (e.g., rod-shaped plant virus particle) that has been modifiedto include an imaging agent is also referred to herein as an imagingnanoparticle.

In general, imaging agents can be conjugated to the plant virus by anysuitable technique, with appropriate consideration of the need forpharmacokinetic stability and reduced overall toxicity to the patient.The term “conjugating” when made in reference to an agent and a plantvirus particle as used herein means covalently linking the agent to thevirus subject to the single limitation that the nature and size of theagent and the site at which it is covalently linked to the virusparticle do not interfere with the biodistribution of the modifiedvirus. The imaging agent can be linked to the interior or the exteriorof the virus, while in some embodiments the imaging agent is linked toboth the interior and the exterior of the virus. The location of theimaging agent on the interior or exterior is governed by the amino acidsof the viral coat protein that are selected as target linking sites. Theinterior surface of the virus particle is the inward-facing side of thevirus particle, which typically faces the nucleic acid within the virusparticle. The exterior surface of the virus particle is the side of thevirus particle facing the environment outside of the virus particle.

The imaging agent(s) can be coupled to a plant virus particle eitherdirectly or indirectly (e.g. via a linker group). In some embodiments,the agent is directly attached to a functional group capable of reactingwith the agent. For example, viral coat proteins include lysines thathave a free amino group that can be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide).Viral coat proteins also contain glutamic and aspartic acids. Thecarboxylate groups of these amino acids also present attractive targetsfor functionalization using carbodiimide activated linker molecules;cysteines can also be present which facilitate chemical coupling viathiol-selective chemistry (e.g., maleimide-activated compounds).Further, viral coat proteins contain tyrosines, which can be modifiedusing diazonium coupling reactions. In addition, genetic modificationcan be applied to introduce any desired functional residue, includingnon-natural amino acids, e.g. alkyne- or azide-functional groups. SeeHermanson, G. T. Bioconjugation Techniques. (Academic Press, 2008) andPokorski, J. K. and N. F. Steinmetz, Mol Pharm 8(1): 29-43 (2011), thedisclosures of which are incorporated herein by reference.

Alternatively, a suitable chemical linker group can be used. A linkergroup can serve to increase the chemical reactivity of a substituent oneither the agent or the virus particle, and thus increase the couplingefficiency. Suitable linkage chemistries include maleimidyl linkers,which can be used to link to thiol groups, isothiocyanate andsuccinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) linkers, which can linkto free amine groups, diazonium which can be used to link to phenol, andamines, which can be used to link with free acids such as carboxylategroups using carbodiimide activation. Useful functional groups arepresent on viral coat proteins based on the particular amino acidspresent, and additional groups can be designed into recombinant viralcoat proteins. It will be evident to those skilled in the art that avariety of bifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), can be employed as a linker group.Coupling can be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues.

Other types of linking chemistries are also available. For example,methods for conjugating polysaccharides to peptides are exemplified by,but not limited to coupling via alpha- or epsilon-amino groups toNaIO₄-activated oligosaccharide (Bocher et al., J. Immunol. Methods 27,191-202 (1997)), using squaric acid diester(1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent (Tietze et al.Bioconjug Chem. 2:148-153 (1991)), coupling via a peptide linker whereinthe polysaccharide has a reducing terminal and is free of carboxylgroups (U.S. Pat. No. 5,342,770), and coupling with a synthetic peptidecarrier derived from human heat shock protein hsp65 (U.S. Pat. No.5,736,146). Further methods for conjugating polysaccharides, proteins,and lipids to plant virus peptides are described by U.S. Pat. No.7,666,624.

When attaching lanthanide imaging agents such as gadolinium ions achelating compound is also used. Conjugation of a chelated lanthanideion to a virus particle can decrease its molecular tumbling rate,resulting in an increased ionic relaxivity rate. A number of chelatingcompounds have been developed to increase the coordinated watermolecules for lanthanide ions, which can almost double the relaxivityrate. Examples of effective gadolinium chelating molecules include1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),diethylenetriaminopentacetate (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7-triasacetic acid (DO3A),6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid (AAZTA), and4-carboxyamido-3,2-hydroxypyridinone (HOPA). See Gugliotta et al., Org.Biomol. Chem., 8, 4569 (2010), the disclosure of which is incorporatedherein by reference. Bifunctional chelating agents includingN-hydroxysuccinimide/isothiocyanates, amine, maleimide, and azidechemical linkers can be used for conjugation to amines, carboxylaticacids, thiols, and alkynes.

In some embodiments, more than one type of imaging agent can be attachedto a plant virus particle. For example, a plant virus particle can bemade useful as an imaging agent for two or more different visualizationtechniques. In further embodiments, differences in the linking sitesavailable on the outside surface (i.e., exterior) and inside channel(i.e., interior) of the virus particle can be used to provide a virusparticle with different imaging agents on the inside and outside of thevirus particle. For example, the virus particle can have a first imagingagent on the inside of the particle, and a second, different imagingagent on the outside of the virus particle. The different linking sitesallow different linking chemistries to be used for the interior andexterior portions of the virus particle. In further embodiments, ratherthan including a different imaging agent, different linking sites can beused to attach a targeting moiety to the virus particle.

The number of imaging agents that can be loaded onto the virus particledepends on the number of attachment sites available and the chemistriesemployed to link the agents to the virus particle. In some embodiments,each virus particle is loaded with about 500 agent molecules. In furtherembodiments, each virus particle is loaded with at least about 1,000,1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or at least about 5,000imaging agent molecules.

Targeting Moieties

In some embodiments, a targeting moiety can also be attached to theplant virus particle. By “targeting moiety” herein is meant a functionalgroup which serves to target or direct the virus particle to aparticular location, cell type, diseased tissue, or association. Ingeneral, the targeting moiety is directed against a target molecule.Thus, for example, antibodies, cell surface receptor ligands andhormones, lipids, sugars and dextrans, alcohols, bile acids, fattyacids, amino acids, peptides and nucleic acids may all be attached tolocalize or target the virus particle to a particular site. In someembodiments, the targeting moiety allows targeting of the plant virusparticles of the invention to a particular tissue or cell type. Forexample, in some embodiments, the targeting moiety specifically binds toan immune cell. Preferably, the targeting moiety is linked to theexterior surface of the virus to provide easier access to the targetmolecule.

In some embodiments, the targeting moiety is a peptide. For example,chemotactic peptides have been used to image tissue injury andinflammation, particularly by bacterial infection; see WO 97/14443,hereby expressly incorporated by reference in its entirety. Anotherexample, are peptides specific to fibrin or vascular cell adhesionmolecules to direct the imaging probe to sites of inflammation, such asan atherosclerotic plaque. In other embodiments, the targeting moiety isan antibody. The term “antibody” includes antibody fragments, as areknown in the art, including Fab Fab₂, single chain antibodies (Fv forexample), chimeric antibodies, etc., either produced by the modificationof whole antibodies or those synthesized de novo using recombinant DNAtechnologies. As is known to those skilled in the art, antibodiesspecifically bind to a particular antigen. In further embodiments, theantibody targeting moieties of the invention are humanized antibodies orhuman antibodies. Humanized forms of non-human (e.g., murine) antibodiesare chimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin.

In some embodiments, the antibody is directed against a cell-surfacemarker on a diseased cell such as a cancer cell; that is, the targetmolecule is a cell surface molecule. As is known in the art, there are awide variety of antibodies known to be differentially expressed on tumorcells, including, but not limited to, HER2. Examples of physiologicallyrelevant carbohydrates may be used as cell-surface markers include, butare not limited to, antibodies against markers for breast cancer (CA15-3, CA 549, CA 27.29), mucin-like carcinoma associated antigen (MCA),ovarian cancer (CA125), pancreatic cancer (DE-PAN-2), and colorectal andpancreatic cancer (CA 19, CA 50, CA242).

In some embodiments, the targeting moiety is all or a portion (e.g. abinding portion) of a ligand for a cell surface receptor. Suitableligands include, but are not limited to, all or a functional portion ofthe ligands that bind to a cell surface receptor selected from the groupconsisting of insulin receptor (insulin), insulin-like growth factorreceptor (including both IGF-1 and IGF-2), growth hormone receptor,glucose transporters (particularly GLUT 4 receptor), transferrinreceptor (transferrin), epidermal growth factor receptor (EGF), lowdensity lipoprotein receptor, high density lipoprotein receptor, leptinreceptor, estrogen receptor (estrogen); interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor (VEGF), PDGF receptor (PDGF), transforming growth factorreceptor (including TGF-α and TGF-β), EPO receptor (EPO), TPO receptor(TPO), ciliary neurotrophic factor receptor, prolactin receptor, andT-cell receptors. Receptor ligands include ligands that bind toreceptors such as cell surface receptors, which include hormones,lipids, proteins, glycoproteins, signal transducers, growth factors,cytokines, and others.

In some embodiments, the targeting moiety binds specifically to animmune cell. Examples of immune cells include neutrophiol, monocytes,macrophages, dendritic cells, natural killer cells, T-cells, andB-cells. A preferred immune cell is the macrophage. A wide variety ofcell surface markers that can be used as the target for targetingmoieties are known to those skilled in the art.

Imaging a Tissue Region

An additional aspect of the present invention provides a method ofgenerating an image of a tissue region of a subject. The method includesadministering to the subject a diagnostically effective amount of animaging nanoparticle, comprising a plant virus particle having aninterior surface and an exterior surface, an imaging agent that islinked to the interior and/or exterior surface, and a layer ofbiocompatible mineral coated over the exterior surface, and generatingan image of the tissue region of the subject to which the imagingnanoparticle has been distributed. The imaging nanoparticle can includeany of the specific features described herein.

In some embodiments, the imaging nanoparticle is used to target tissuein a subject without the use of a targeting moiety based on the abilityof plant virus particles to preferentially accumulate in certaintissues. In particular, the plant virus particles have been shown topreferentially accumulate in diseased tissue, such as cancer tissue orinflamed tissue (e.g., atherosclerotic blood vessels). While notintending to be bound by theory, it appears that plant virus particles(e.g., rod-shaped plant virus particles) are taken up by bloodcomponents such as macrophage cells of the immune system, whichsubsequently accumulate in diseased tissue (e.g., a tumor oratherosclerotic blood vessel), thereby delivering the plant virus tocells at the disease site.

A tumor is an abnormal mass of tissue as a result of abnormal growth ordivision of cells caused by cancer. Tumors can occur in a variety ofdifferent types of tissue such as the breast, lung, brain, liver kidney,colon, and prostate, can be malignant or benign, and generally vary insize from about 1 cm to about 5 cm.

Magnetic resonance angiography (MRA) is a type of MRI that generatespictures of blood vessels (e.g., arteries) to evaluate them for stenosis(abnormal narrowing) or aneurysms (vessel wall dilatations, at risk ofrupture). MRA can be used to evaluate the arteries of the neck andbrain, the thoracic and abdominal aorta, the renal arteries, and thelegs. Imaging nanoparticles can be used to facilitate conducing MRA ofblood vessels for various uses, including evaluation of the possibledevelopment of atherosclerosis. Atherosclerosis is a chronicinflammatory response in the walls of arteries, caused largely by theaccumulation of macrophages and white blood cells and promoted bylow-density lipoproteins (LDL, plasma proteins that carry cholesteroland triglycerides) without adequate removal of fats and cholesterol fromthe macrophages by functional high-density lipoproteins (HDL). It iscommonly referred to as a hardening or furring of the arteries, and iscaused by the formation of multiple plaques within the arteries, whichcan be detected by MRA.

In order to generate an image of the tissue region, it is necessary foran effective amount of imaging agent to reach the tissue region ofinterest, but it is not necessary that the imaging agent be localized inthis region alone. However, in some embodiments, the imagingnanoparticles are targeted or administered locally such that they arepresent primarily in the tissue region of interest. In some embodiments,SNPs formed from rod-shaped plant virus particles are used. Examples ofimages include two-dimensional cross-sectional views and threedimensional images. In some embodiments, a computer is used to analyzethe data generated by the imaging agents in order to generate a visualimage. The plant virus particles can include any of the virus particlesdescribed herein, such as Virgaviridae virus particles and tobaccomosaic virus particles. The tissue region can be an organ of a subjectsuch as the heart, lungs, or blood vessels. In other embodiments, thetissue region can be diseased tissue, or tissue that is suspected ofbeing diseased, such as a tumor or atherosclerotic tissue. Examples ofimaging methods include fluoroscopy, computed tomography, positiveemission tomography, and magnetic resonance imaging.

Means of detecting labels in order to generate an image are well knownto those of skill in the art. Thus, for example, where the label is aradioactive label, means for detection include a scintillation counteror photographic film as in autoradiography. Where the label is afluorescent label, it may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence. The fluorescence may be detected visually, by means ofphotographic film, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Finally simplecolorimetric labels may be detected simply by observing the colorassociated with the label.

“Computed tomography (CT)” refers to a diagnostic imaging tool thatcomputes multiple x-ray cross sections to produce a cross-sectional viewof the vascular system, organs, bones, and tissues. “Positive emissionstomography (PET)” refers to a diagnostic imaging tool in which thepatient receives a radioactive isotopes by injection or ingestion whichthen computes multiple x-ray cross sections to produce a cross-sectionalview of the vascular system, organs, bones, and tissues to image theradioactive tracer. These radioactive isotopes are bound to compounds ordrugs that are injected into the body and enable study of the physiologyof normal and abnormal tissues. “Magnetic resonance imaging (MRI)”refers to a diagnostic imaging tool using magnetic fields and radiowavesto produce a cross-sectional view of the body including the vascularsystem, organs, bones, and tissues. Suitable imaging agents should beused that will help generate an image of a tissue region in the contextof the imaging technique being used. For example, when using magneticresonance imaging, a suitable imaging agent is a chelated lanthanide.

In some embodiments, the imaging nanoparticles of the present inventionare used for MRI. MRI provides a good contrast between the differentsoft tissues of the body, which makes it especially useful in imagingthe brain, muscles, the heart, and cancers compared with other medicalimaging techniques such as computed tomography or X-rays. An MRI scanneris a device in which the subject lies within a large, powerful magnetwhere the magnetic field is used to align the magnetization of someatomic nuclei in the body, and radio frequency magnetic fields areapplied to systematically alter the alignment of this magnetization.This causes the nuclei to produce a rotating magnetic field detectableby the scanner and this information is recorded to construct an image ofa tissue region. Magnetic field gradients cause nuclei at differentlocations to process at different speeds, allowing spatial informationto be recovered using Fourier analysis of the measured signal. By usinggradients in different directions, 2D images or 3D volumes can beobtained in any arbitrary orientation

Various different types of MRI scans can be conducted, includingT₁-weighted MRI, T₂-weighted MRI, and spin density weighted MRI. In someembodiments, the viral imaging agents of the invention are used ascontrast agents to facilitate a T₁-weighted MRI scan. T₁-weighted scansrefer to a set of standard scans that depict differences in thespin-lattice (or T₁) relaxation time of various tissues within the body.T₁ weighted images can be acquired using either spin echo orgradient-echo sequences. T₁-weighted contrast can be increased with theapplication of an inversion recovery RF pulse. Gradient-echo basedT₁-weighted sequences can be acquired very rapidly because of theirability to use short inter-pulse repetition times (TR).

Immunogenicity of Imaging Nanoparticles

Administering viral particles to a subject is known to sometimesgenerate an immune response. An “immune response” refers to theconcerted action of lymphocytes, antigen presenting cells, phagocyticcells, granulocytes, and soluble macromolecules produced by the abovecells or the liver (including antibodies, cytokines, and complement)that results in selective damage to, destruction of, or elimination fromthe human body of cancerous cells, metastatic tumor cells, invadingpathogens, cells or tissues infected with pathogens, or, in cases ofautoimmunity or pathological inflammation, normal human cells ortissues. Components of an immune response can be detected in vitro byvarious methods that are well known to those of ordinary skill in theart.

An advantage of the imaging nanoparticles of the present invention isthat they exhibit decreased immunogenicity as a result of including alayer of biocompatible mineral. As noted above, viral particles areknown to often induce an immune response when administered to a subject.Coating the plant virus particles with a biocompatible mineral decreasesor in some cases eliminates this immune response. In some embodiments,the biocompatible mineral can decrease the immune response by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or in some cases by 100%.While not intending to be bound by theory, the decreased immunogenicityexhibited by viral particles coated with a biocompatible mineral mayoccur as a result of a decreased ability for the viral particles to berecognized by antibodies.

Administration and Formulation of Imaging Nanoparticles

In some embodiments, the imaging nanoparticles are administered togetherwith a pharmaceutically acceptable carrier to provide a pharmaceuticalformulation. Pharmaceutically acceptable carriers enable the imagingnanoparticles to be delivered to the subject in an effective mannerwhile minimizing side effects, and can include a variety of diluents orexcipients known to those of ordinary skill in the art. Formulationsinclude, but are not limited to, those suitable for oral, rectal,vaginal, topical, nasal, ophthalmic, or parental (includingsubcutaneous, intramuscular, intraperitoneal, intratumoral, andintravenous) administration. For example, for parenteral administration,isotonic saline is preferred. For topical administration, a cream,including a carrier such as dimethylsulfoxide (DMSO), or other agentstypically found in topical creams that do not block or inhibit activityof the compound, can be used. Other suitable carriers include, but arenot limited to, distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, alcohol, dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants, or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the imagingnanoparticle into association with a pharmaceutically acceptable carrierthat constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing theimaging nanoparticle into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into the desired formulations.

Useful dosages of the active agents can be determined by comparing theirin vitro activity and the in vivo activity in animal models. Methods forextrapolation of effective dosages in mice, and other animals, to humansare known in the art; for example, see U.S. Pat. No. 4,938,949. Anamount adequate to accomplish imaging is a diagnostically effectiveamount. Effective doses of the imaging nanoparticle vary depending uponmany different factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered.

For administration for imaging in a mammalian subject utilizing animaging nanoparticle, the dosage of the imaging agent ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. A suitable amount of virusparticle is used to provide the desired dosage. An exemplary treatmentregime entails administration once per every two weeks or once a monthor once every 3 to 6 months. The imaging nanoparticle can beadministered on multiple occasions. Alternatively, the imagingnanoparticle can be administered as a sustained release formulation, inwhich case less frequent administration is required.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes).

The present invention is illustrated by the following example. It is tobe understood that the particular example, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLE Example 1: Silica-Coated Gd(DOTA)-Loaded Protein NanoparticlesEnable Magnetic Resonance Imaging of Macrophages

The molecular imaging of in vivo targets allows non-invasive diseasediagnosis. Nanoparticles offer a promising platform for molecularimaging because they can deliver large payloads of imaging reagents tothe site of disease. Magnetic resonance imaging (MRI) is often preferredfor clinical diagnosis because it uses non-ionizing radiation and offersboth high spatial resolution and excellent penetration. The inventorshave explored the use of plant viruses as the basis of for MRI contrastreagents, specifically Tobacco mosaic virus (TMV), which can assemble toform either stiff rods or spheres. TMV particles were loaded withparamagnetic Gd ions, increasing the ionic relaxivity compared to freeGd ions. The loaded TMV particles were then coated with silicamaintaining high relaxivities. Interestingly, it was found that whenGd(DOTA) was loaded into the interior channel of TMV and the exteriorwas coated with silica, the T1 relaxivities increased by three-fold from10.9 mM⁻¹ s⁻¹ to 29.7 mM⁻¹ s⁻¹ at 60 MHz compared to uncoated Gd-loadedTMV. To test the performance of the contrast agents in a biologicalsetting, the inventors focused on interactions with macrophages becausethe active or passive targeting of immune cells is a popular strategy toinvestigate the cellular components involved in disease progressionassociated with inflammation. In vitro assays and phantom MRIexperiments indicate efficient targeting and imaging of macrophages,enhanced contrast-to-noise ratio was observed by shape-engineering(SNP>TMV) and silica-coating (Si-TMV/SNP>TMV/SNP). Because plant virusesare in the food chain, antibodies may be prevalent in the population. Itwas therefore investigated whether the silica-coating could preventantibody recognition; indeed the data indicate that mineralization canbe used as a stealth coating option to reduce clearance. The inventorstherefore conclude that the silica-coated protein-based contrast agentmay provide an interesting candidate material for further investigationfor in vivo delineation of disease through macrophage imaging.

Results and Discussion

The nanoparticles were based on a mutant of TMV (S152K, TMVlys) thatdisplays a reactive amine-functional lysine group at the solvent-exposedC-terminus of the coat protein. Geiger et al., Nanoscale. 2013;5(9):3808-3816. TMVlys was produced in Nicotiana benthamiana plants witha yield of 5 mg pure TMVlys particles per gram of infected leafmaterial. TMVlys comprises 2130 identical coat proteins arrangedhelically into a 300-nm soft-matter rod, 18 nm in diameter with a 4-nminternal channel. TMVlys was modified with paramagnetic Gd^(III)chelated to azido-monoamide-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA-azide) to yield the MRI contrast-enhancement agent. Bruckman etal., Journal of Materials Chemistry B. 2013; 1(10):1482. Thebioconjugation of Gd(DOTA) to the internal and external surfaces ofTMVlys is described in detail in the Supporting Information and FIG. 5 .Briefly, terminal alkyne functionality was provided by modifying thetyrosine (TYR139) or glutamic acid (GLU 97/106) residues. Bruckman etal., ACS Biomater Sci Eng., 2015; 1(1):13-8. Gd(DOTA) was attached tothe terminal alkynes using a copper-catalyzed azide-alkyne cycloadditionreaction, forming internal Gd(DOTA) TMVlys (iGd-TMV) or externalGd(DOTA) TMVlys (eGd-TMV). TMVlys-based spherical nanoparticles (SNPs)were produced by heating iGd-TMV to 96° C. for 60 s using a PCRthermocycler, forming Gd-SNP. Gd(DOTA) labeling efficiency (FIG. 2A) wascharacterized by inductively-coupled plasma optical emissionspectroscopy (ICP-OES) and matrix assisted laser desorption-ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) as shown in FIG. 6 . TEMimaging was used to confirm the structural integrity of the TMV rods andspheres after chemical modification (FIG. 1B). The TMV rods weremodified with an average of 1000 Gd(DOTA) molecules per particle in boththe internal and external labeling configurations. Similar loadingdensity was achieved for SNPs, with 976 Gd(DOTA) per 2130 coat proteins(based on absorbance) on a particle with a diameter of ˜75 nm and adensity of 1.43 g/cm³, compared to 1.31 g/cm³ for the TMV rods. Dobrovet al., Journal of Biomolecular Structure and Dynamics. 2014;32(5):701-8. This corresponds to an estimated 7074 coat proteins persphere, yielding just over 3000 Gd(DOTA) labels per SNP.

Silica coatings were introduced as described, with modifications. Stoberet al., Journal of Colloid and Interface Science. 1968; 26(1):62-69.Briefly, 1 ml tetraethyl orthosilicate (TEOS) as a 10% v/v stock inethanol was mixed with 4 ml 5 M NH₄OH and 1 ml of modified TMVlys orSNPs (1 mg/ml in water) and diluted to 20 ml with ice-cold ethanol. Thereaction was allowed to proceed overnight. The resulting Si coat was ˜65nm deep, increasing the thickness of the nanoparticles to ˜150 nm asshown by TEM and SEM (FIG. 1B). The TMVlys mutant allowed the formationof a higher-quality silica coating compared to native TMV, because thepositive charge of the solvent-exposed amine groups favored thenucleation of silica catalyzed by TEOS. The presence of silica wasconfirmed by TEM using electron dispersion spectroscopy (EDS) as shownin FIG. 7 . Silica mineralization presented the following challenges:First, the proportion of ethanol must be ˜90% after all reactants havebeen added because lower concentrations increase the abundance of freesilica particles, and high concentrations result in much thinnercoatings. Additionally, salt must be removed from the TMV solutionbefore mineralization. Sonication reduced aggregation and improved thedispersion of the mineralized particles. This is consistent withprevious reports describing the silica mineralization of TMV (Fowler etal., Advanced Materials, 2001; 13(16):1266-9), fd phage (Zhang Z,Buitenhuis J., Small. 2007; 3(3):424-8), and other nanoparticles (Liu etal., Advanced Materials. 2014; 27(3):479-97). Silica is ideal as acoating material due to its biocompatibility, ease of surfacefunctionalization, versatility, and stability. Tamba et al., EuropeanJournal of Pharmaceutical Sciences. 2015; 71:46-55.

Next, the longitudinal proton relaxivity of Gd(DOTA)-modified TMV rodsand spheres (SNPs) was measured using a standard inversion recoverysequence on a 60 MHz relaxometer (Bruker) at 37° C. (FIG. 2 ).Previously, the inventors found that TMV externally modified withGd(DOTA) had a higher ionic relaxivity than internally-modifiedparticles due to increased stiffness of the tyrosine residue and betterbulk water accessibility. Bruckman et al., Journal of MaterialsChemistry B. 2013; 1(10):1482. Overall, the same trend was observed inthe present study.

Throughout the investigation, some batch-to-batch variations in theionic relaxivity were noted when TMV samples were compared withdifferent Gd(DOTA) loading rates. Therefore, the inventors set out todetermine whether the Gd(DOTA) density affected the ionic relaxivity ofthe particles. TMV rods with Gd(DOTA) loads ranging from 429 to 1477 Gdper particle were prepared and the ionic relaxivities were determined(FIG. 8 ). An inverse correlation between ionic relaxivity and Gd(DOTA)density was found. Although the per-particle relaxivity increased athigher Gd density, the ionic relaxivity decreased at higher Gd density.If the Gd(DOTA) ions are distributed in a statistically random manner,the lower-density formulation may offer greater inter-Gd(DOTA) spacingand therefore may increase the number of water molecules showinginteractions at any given time. Others have shown that greater spacingbetween Gd ions (lower density) can increase overall relaxivity byincreasing the transverse electronic relaxivity of the ions.Interactions between nearby paramagnetic centers increase the electronicrelaxation of the electron spins of Gd ions, thereby reducing the waterrelaxivity. Nicolle et al., Magnetic Resonance in Chemistry. 2003;41(10):794-9. For example, micelles fully loaded (100%) with chelated Gdwere able to achieve relaxivities of 30.0 mM⁻¹ s⁻¹ at 20 MHz, whereasmicelles loaded with 98% Y and 2% Gd achieved a relaxivity of 41.4 mM⁻¹s⁻¹ at 20 MHz. Gianolio et al., Chemistry—A European Journal. 2007;13(20):5785-97. Alternatively, if one considers iGd-TMV and assumes thelabeling density increases at the open ends, this would allow for moreefficient water exchange with the bulk water surrounding the TMV rodwithout limiting water exchange in the 4-nm internal channel. Indeed,similar trends have been reported with mesoporous silica nanoparticleslabeled with Gd when the entrance to the pores is compared to the entirestructure. Davis et al., Journal of Materials Chemistry. 2012;22(43):22848-50.

Next, the relaxivities of mineralized TMV rods and SNPs versus theirnative counterparts were compared. The ionic and per particlerelaxivities remained consistent for eGd-TMV (23.5 vs 24.8 mM⁻¹ s⁻¹) andSNP (17.7 vs 16.5 mM⁻¹ s⁻¹) following silica coating (FIG. 2 ). Silicamineralization alone did not change the relaxivity compared toconcentration-matched unlabeled TMVlys particles (FIG. 2F). In starkcontrast, a nearly three-fold increase in relaxivity was observed formineralized versus native iGd-TMV particles, i.e. there was an increasefrom 10.9 to 29.7 mM⁻¹ s⁻¹ at 60 MHz which is presented as a bar chart(FIG. 2B) and in the form of relaxivity curves (FIG. 2C-F).

Two factors may promote this increased relaxivity. First, mineralizationaround the TMV scaffold creates a dense surface coating that may trapbulk water inside the 4-nm channel resulting in differential water fluxconnecting the bulk surrounding water and the bulk internal water,maintaining and increasing the molecular interactions between Gdmolecules and internal bulk water, therefore increasing the relaxivityof iGd-TMV but not eGd-TMV. Given that the silica-coated eGd-TMV andGd-SNP formulations exhibited T₁ values comparable to their native formssuggests that mesoporous silica is formed enabling water exchangethrough the silica coat. High-resolution TEM confirms the formation ofmesoporous silica shells on the TMV rods and spheres (FIG. 9 ). Second,particle stiffness increases when the silica coating is applied, andthis may increase the relaxivity by inhibiting the molecular tumbling ofGd(DOTA). Nanoparticle rigidity can be calculated using the Lipari-Szaboapproach, which identifies an order parameter for the local and globalrotations with limiting values 0<S²<1, where 1 is a completely rigidnanoparticle and 0 is a fully independent contrast agent. Verwilst etal., Chemical Society Reviews. 2015; 44(7):1791-1806. Stiffernanoparticles have higher order parameter values (S²) and thereforeyield a higher ionic relaxivity. Botta M, Tei L., European Journal ofInorganic Chemistry. 2012; 2012(12):1945-60.

To test the performance of the contrast agents in a biological setting,the inventors focused on interactions with macrophages because theactive or passive targeting of immune cells is a popular strategy toinvestigate the cellular components involved in disease progression. Forexample, targeting macrophages in cardiovascular disease can provideinsight into the composition of atherosclerotic plaques and mayfacilitate risk stratification. A macrophage-rich and lipid-rich plaquewith a thin fibrous cap may indicate a plaque vulnerable to rupture.Kooi et al., Circulation. 2003; 107(19):2453-8. RAW 264.7 murinemacrophages were therefore used as a model system. Nel et al., NatureMaterials. 2009; 8(7):543-57. The uptake of TMV rods and spheres wastested before and after mineralization by flow cytometry usingfluorescence-labeled particles (FIGS. 3A and 3B) and then tested theGd(DOTA)-labeled formulations in MRI experiments (FIGS. 3C and 3D). Thelabeling strategy and characterization of fluorescent particles isdescribed in the Methods section.

Time-course flow cytometry showed that the silica coating significantlyincreased the number of interactions between macrophages and the TMVnanoparticles, regardless of their shape (FIG. 3A) agreeing withprevious reports showing that macrophages rapidly scavenge silicananoparticles. Zhu et al., Nanoscale. 2014; 6(19):11462-72. Thenon-coated SNPs were much more readily taken up by the macrophages thanTMVlys rods (FIG. 3A). This is consistent with a recent study by theinventors showing that macrophages interact more efficiently with TMVparticles with a low aspect ratio. Shukla et al., Adv Healthc Mater.2015; 4(6):874-882. The high aspect ratio of the elongated stiff rodpromotes immune evasion by inhibiting phagocytosis. This is a well-knownphenomenon in nanomedicine and can be advantageous if cellular ormolecular components other than macrophages are the desired target. Wenet al., Journal of Biological Physics. 2013; 39(2):301-25.

The inventors next investigated whether the Gd-labeled TMV and SNPformulations could be used to detect macrophages in a pre-clinical MRIscanner (FIG. 3C). Briefly, cells were incubated with the different MRIcontrast agents and controls, and were then pelleted by centrifugationand analyzed using a 7.0 T (300 MHz) MRI (Bruker BioSpec 70/30USR).Following multiple scouting scans, a T₁-weighted Multi Slice Multi Echo(MSME) sequence was used with the following parameters: TR/TE=600/8.0ms, 1 mm thickness, four averages, matrix=128×128, field of view=2.98cm. Exported DICOM images were analyzed with the free open softwareOsiriX.

Macrophages were quantified by measuring the contrast-to-noise ratio(CNR) of cell pellets compared to the buffer solution for each well. Itwas found that silica-coated TMV rods and SNPs showed higher CNRs thantheir non-mineralized counterparts (FIG. 3D), which is consistent withthe interactions observed by flow cytometry (FIG. 3B). Additionally,SNPs yielded higher CNRs than TMV rods confirming that SNPs targetmacrophages more efficiently than rods. The experiments werereproducible over a range of cell and particle concentrations (FIG. 10).

Together, these results demonstrate that SNP, Si-TMV and Si-SNPparticles are suitable for the imaging of macrophage-rich diseases. Theinventors have previously shown that targeted rod-shaped TMV particlesare appropriate for MRI applied to vasculature molecular markers inatherosclerotic plaques(14). TMV rods could therefore be combined withSNPs to image molecular markers and macrophages, providing a powerfultool to facilitate risk stratification and the prognosis ofatherosclerosis patients.

Lastly, the inventors set out to determine whether the silica coatingwould protect the TMV protein-based contrast agents from antibodyrecognition. VNPs, much like other protein-based nanoparticles, areprone to elicit the production of VNP-specific antibodies whenintroduced as ‘naked’ versions into the body. Furthermore, earlyresearch has shown that TMV-specific antibodies are prevalent due topresence of TMV in food and cigarettes. Therefore, they investigatedwhether the silica shell would protect TMV (and SNP) from recognition bythe immune system. This is an important requirement in translationalapplications because antibody binding can interfere with targetrecognition and alter the fate of nanoparticle-based MRIcontrast-enhancing reagents, particularly if repeat administration isnecessary.

To determine the ability of the thick silica coating to prevent antibodyrecognition, immunogold staining experiments were carried out in whichTMV, SNP, Si-TMV or Si-SNP were deposited on TEM grids followed by theapplication of TMV-specific antibodies and detection using 10-nm goldimmunoconjugate secondary antibodies (FIG. 4 , see SupportingInformation for details). Accordingly, it was found that the non-coatedSNP and TMV particles were efficiently recognized by the antibodies,whereas the silica-coated TMV and SNP formulations were shielded fromantibody recognition. This was confirmed by testing mixed preparationsof TMV and Si-TMV, which resulted in the specific recognition of thenon-coated TMV particles (FIG. 4E). Silica coatings could therefore beapplied as an alternative to polyethylene glycol (PEG) shielding toavoid antibody recognition. The inventors have previously shown that PEGshielding yields stealth VNPs that are not recognized by antibodies. Leeet al., Acta Biomater. 2015; 19:166-179. Whereas Si is known to improvethe biocompatibility and reduce the toxicity nanoparticles based ongold, iron oxide and quantum dots, this is the first demonstration thatSi can also circumvent immune surveillance.

Conclusion

Rod-shaped and spherical silica-coated TMV nanoparticles loaded withGd(DOTA) were synthesized. Silica-coated contrast agents maintained highrelaxivities, therefore providing a potential candidate material for MRIapplications. Interestingly, it was found that the mineralization of TMVrods labeled internally with Gd(DOTA) increased the ionic relaxivity ofthe particles three-fold compared to non-mineralized particles,potentially reflecting the increased particle stiffness. Medicalrelevance was determined in vitro using the murine macrophage cell lineRAW 264.7. These studies serve as a proof-of-concept; detection andimaging of macrophages may aid diagnosis and prognosis of diseaseassociated with inflammation, such as cardiovascular diseases. Imagingstudies demonstrate increased macrophage targeting as a function ofnanoparticle shape and surface coating with SNP>TMV and Si-coatedSNP/TMV>native SNP/TMV. Lastly, the inventors demonstrate that thesilica-coating effectively reduced antibody binding, which is importantfor the translational development of these MRI contrast agents. Thisversatile mineralization protocol could also be applied to otherplatforms for biological macromolecule cargo delivery to reduceimmunogenicity and may improve MRI contrast relaxivity.

Methods

TMV Manufacturing

Propagation. The TMV lysine mutant S152K was propagated in Nicotianabenthamiana plants and recovered, with a yield of 5 mg TMV per graminfected leaf material, using established extraction methods. M. A.Bruckman and N. F. Steinmetz, Methods in molecular biology, 2014, 1108,173-185.

The concentration of TMV in plant extracts was determined by UV/visspectroscopy (ε_(260nm)=3.0 mg⁻¹ mL cm⁻¹) and virus integrity wasverified by TEM and SEM imaging.

Bioconjugation. Gd(DOTA)-labeled particles were prepared as previouslydescribed. Bruckman et al., Journal of Materials Chemistry B, 2013, 1,1482. Briefly, the TMV external surface was modified with a diazoniumsalt generated from 3-ethynylaniline (25 molar equivalents (eq), pH=8.5,30 min) to incorporate a terminal alkyne. Similarly, the internalchannel was modified in the same way by mixing propargylamine (50 eq)with ethyldimethylpropylcarbodiimide (EDC, 100 eq) andn-hydroxybenzotriazole (HOBt, 50 eq) for 24 h. Gd was chelated toazido-mono amide-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraaceticacid (DOTA-azide, Macrocyclics) as previously described. Bruckman etal., Journal of Materials Chemistry B, 2013, 1, 1482. Briefly, a 1:1mixture of GdCl₃ and DOTA-azide in water was mixed at room temperaturefor 3 days while maintaining the pH at 6-7 (tested using pH paper).After 3 days, the pH was increased to 9-10 and the precipitate wasremoved by centrifugation. Efficient conjugation of Gd(DOTA) azide toTMV terminal alkyne groups was accomplished by copper-catalyzedazide-alkyne cycloaddition (CuAAC) to form TMV particles withGd-conjugated externally (eGd-TMV) or internally (iGd-TMV).Alkyne-labeled TMV (2 mg/mL) in 0.1 M potassium phosphate buffer (pH7.0) was mixed with Gd(DOTA) azide (2 eq), aminoguanidine (2 mM),ascorbic acid (2 mM) and copper sulfate (1 mM) for 30 min on ice. Thereaction mix was purified by ultracentrifugation in a 10-40% sucrosegradient, and analyzed by TEM and MALDI-TOF MS. For flow cytometryexperiments, sulfo-Cy5 azide (Lumiprobe) was used in place of Gd(DOTA)azide to synthesize internally-labeled Cy5-TMV.

Thermal transition to SNP. The standard protocol for thermal transitionfrom native TMV rods to SNPs involves heating a sample of TMV rods (0.3mg mL⁻¹) for 60 s at 96° C. in a Peltier thermal cycler. SNPs are thenrecovered by centrifugation at 42,000 rpm for 2 h.

Mineralization. One mL of TMV or SNP particles (1 mg mL⁻¹) was added to18 mL ethanol on ice and mixed in 1 mL TEOS (10% (v/v) in ethanol) and 4mL 5 M NH₄OH, alternating every 15 min for 1 h. The reaction wasincubated at 4° C. for 18 h followed by centrifugation at 3000 rpm for20 min. The samples were washed with water and centrifuged againfollowed by overnight dialysis against water.

Particle Characterization

Inductively-coupled plasma optical emission spectroscopy (ICP-OES). TheGd loading of modified TMVs or SNPs before and after mineralization wasdetermined by ICP-OES. Samples were diluted to give a proteinconcentration of 0.1 mg mL⁻¹ in pure water and analyzed immediately.

Relaxivity. The ionic relaxivity of the Gd(DOTA)-loaded TMVs and SNPswas tested using a Bruker Minispec mq60 relaxometer at 60 MHz. Astandard inversion recovery sequence was used to determine the T₁.

Transmission electron microscopy (TEM). Drops of TMV rods or SNPs beforeand after mineralization (0.2 mg mL⁻¹ in 5 mL deionized water) wereplaced on copper TEM grids, adsorbed for 5 min, washed with deionizedwater, and negatively stained with 2% (w/v) uranyl acetate for 2 min.Samples were examined by energy dispersive X-ray spectroscopy using aZeiss Libra 200FE transmission electron microscope operated at 200 kV.

Scanning electron microscopy (SEM). Samples were dried onto siliconwafers and then mounted on the surface of an aluminum pin stub usingdouble-sided adhesive carbon discs (Agar Scientific). The stubs werethen sputter-coated with gold (or palladium) in a high-resolutionsputter coater (Agar Scientific, Ltd.) and transferred to a Hitachi 4500scanning electron microscope.

Matrix assisted laser desorption-ionization time-of-flight massspectrometry (MALDI-TOF MS). Native and modified TMV particles (10-20 mgin 24 μL water) were denatured by adding 6 μL 6 M guanidinehydrochloride and mixing for 5 min at room temperature. Denaturedproteins were spotted onto an MTP 384 massive target plate using mC18Zip-Tips (Millipore). MALDI-MS analysis was carried out using a BrukerUltra-Flex I TOF/TOF mass spectrometer.

Particle Testing In Vitro

Cell culture. RAW 264.7 cells (ATCC) were maintained in Dubelco'sminimal essential medium (DMEM) at 37° C. in a 5% CO₂ humidifiedatmosphere. The medium was supplemented with 10% (v/v) heat-inactivatedfetal bovine serum (FBS), 1% (v/v) L-glutamine, and 1% (v/v)penicillin-streptomycin. All reagents were obtained from Gibco.

Flow cytometry. RAW 264.7 cells (500,000 cells in 200 μL DMEM per well)were added to an untreated 96-well v-bottom plate. The iGd-TMV, Gd-SNP,iGd-TMV-Si, and Gd-SNP-Si particles were added at a concentration of100,000 particles/cell in triplicate and incubated for 1, 3, and 8 h at37° C. in a 5% CO₂ humidified atmosphere. Following incubation, thecells were pelleted at 500×g for 4 min. The supernatant was removed, andthe cells were resuspended in FACS buffer (1 mM EDTA, 1% (v/v) FBS, and25 mM HEPES, pH 7.0 in Ca²⁺ and Mg²⁺ free PBS). This washing step wascarried out three times. The cells were then fixed in 2% (v/v)paraformaldehyde in FACS buffer for 10 min at room temperature andwashed another three times. Analysis was carried out using the BD LSR IIflow cytometer, and 10,000 events per sample were collected.

Magnetic resonance imaging (MRI) of cell pellets. RAW cells (5×10⁶ cellsin 1 mL DMEM per tube) were added to untreated 1.5 mL Eppendorf tubes.The iGd-TMV, Gd-SNP, iGd-TMV-Si, and Gd-SNP-Si particles were added at aconcentration of 1×10⁶ particles/cell and incubated for 8 h at 37° C. ina 5% CO₂ humidified atmosphere. Following incubation, the cells werepelleted at 500×g for 4 min. The supernatant was removed, and the cellswere resuspended in FACS buffer. This washing step was carried out threetimes. The cells were then fixed in 2% (v/v) paraformaldehyde in FACSbuffer for 10 min at room temperature and washed another three times.Cells were pelleted in a custom 384 well plate and analysis was carriedout using a pre-clinical 7.0 T (300 MHz) MRI (Bruker BioSpec 70/30USR).Following multiple scouting scans, a T₁-weighted Multi Slice Multi Echo(MSME) sequence was used with the following parameters: TR/TE=600/8.0ms, 1 mm thickness, four averages, matrix=128×128, field of view=2.98cm. Exported DICOM images were analyzed with the free open softwareOsiriX.

Contrast-to-noise (CNR) calculations. The contrast-to-noise ratio wasdetermined by dividing the mean intensity of the cell pellet area overthe mean intensity of the buffer area.

Immunogold labeling. TMV samples were dried on TEM grids, washed with 10mM sodium phosphate buffer pH 7.0 and floated on a drop of 1% (w/v)bovine serum albumin (BSA) in Tris-buffered saline pH 7.4 plus 0.1%(v/v) Tween-20 (TBST) for 30 min. Samples were equilibrated with 0.1%BSA for 5 min before binding for 1 h with a rabbit anti-TMV antibody (10μg mL⁻¹ in 0.1% BSA). The grids were then washed three times with 0.1%(w/v) BSA before binding with goat anti-rabbit secondary antibodiesconjugated to 10-nm gold nanoparticles for 2 h. The grids were thenwashed in phosphate-buffered saline pH 7.4 plus 0.01% (v/v) Tween-20(PBST), then water, prior to staining with 2% (v/v) uranyl acetate for 1min. The grids were imaged by TEM.

The complete disclosure of all patents, patent applications, andpublications, and electronically available materials cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. In particular,the inventors are not bound by theories described herein. The inventionis not limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

What is claimed is:
 1. An imaging nanoparticle, comprising a tobaccomosaic virus particle having an interior surface and an exteriorsurface, a chelated lanthanide imaging agent that is linked to theexterior surface, and a layer of silica coated over the exteriorsurface, wherein the tobacco mosaic virus particle is selected from thegroup consisting of iGd-TMV-Si, eGd-TMV, eGd-TMV-Si, Gd-SNP, andGd-SNP-Si, and wherein the tobacco mosaic virus particle has arelaxivity of greater than about 25,000 mM⁻¹S⁻¹ per particle.
 2. Theimaging nanoparticle of claim 1, wherein a targeting moiety is linked tothe exterior surface of the virus particle.
 3. The imaging nanoparticleof claim 2, wherein the targeting moiety binds specifically to an immunecell.
 4. The imaging nanoparticle of claim 1, wherein at least about 500imaging agent molecules are linked to the virus particle.
 5. The methodof claim 1, wherein the silica maintains the ionic relaxivity of theparticles compared to the ionic relaxivity of a non-siliconizedparticle.
 6. The method of claim 1, wherein the silica reduces theimmunogenicity of the particles when administered to a subject comparedto a non-siliconized particle.
 7. A method of generating an image of atissue region of a subject, by administering to the subject adiagnostically effective amount of an imaging nanoparticle, comprising atobacco mosaic virus particle having an interior surface and an exteriorsurface, a chelated lanthanide imaging agent that is linked to theexterior surface, and a layer of silica coated over the exteriorsurface, wherein the tobacco mosaic virus particle is selected from thegroup consisting of iGd-TMV-Si, eGd-TMV, eGd-TMV-Si, Gd-SNP, andGd-SNP-Si, and wherein the tobacco mosaic virus particle has arelaxivity of greater than about 25,000 mM⁻¹S⁻¹ per particle, andgenerating an image of the tissue region of the subject to which theimaging nanoparticle has been distributed.
 8. The method of claim 7,wherein the method of generating an image is magnetic resonance imaging.9. The method of claim 7, wherein the imaging nanoparticle furthercomprises a targeting moiety is linked to the exterior surface of thevirus particle.
 10. The method of claim 9, wherein the targeting moietyspecifically binds to an immune cell.
 11. The method of claim 7, whereinthe tissue region includes a blood vessel.
 12. The method of claim 7,wherein the silica maintains the ionic relaxivity of the particlescompared to the ionic relaxivity of a non-mineralized particle.
 13. Themethod of claim 7, wherein the silicon reduces the immunogenicity of theparticles when administered to the subject compared to a non-siliconizedparticle.